Sharpe et al. J Transl Med (2020) 18:374 https://doi.org/10.1186/s12967-020-02518-2 Journal of Translational Medicine

RESEARCH Open Access Cannabis, a cause for anxiety? A critical appraisal of the anxiogenic and anxiolytic properties Lara Sharpe1, Justin Sinclair1, Andrew Kramer1, Michael de Manincor1 and Jerome Sarris1,2*

Abstract Background: Cannabis has been documented for use in alleviating anxiety. However, certain research has also shown that it can produce feelings of anxiety, panic, paranoia and psychosis. In humans, Δ9-tetrahydrocannabinol (THC) has been associated with an anxiogenic response, while anxiolytic activity has been attributed mainly to can- nabidiol (CBD). In animal studies, the efects of THC are highly dose-dependent, and biphasic efects of cannabinoids on anxiety-related responses have been extensively documented. A more precise assessment is required of both the anxiolytic and anxiogenic potentials of phytocannabinoids, with an aim towards the development of the ‘holy grail’ in cannabis research, a medicinally-active formulation which may assist in the treatment of anxiety or mood disorders without eliciting any anxiogenic efects. Objectives: To systematically review studies assessing cannabinoid interventions (e.g. THC or CBD or whole cannabis interventions) both in animals and humans, as well as recent epidemiological studies reporting on anxiolytic or anxi- ogenic efects from cannabis consumption. Method: The articles selected for this review were identifed up to January 2020 through searches in the electronic databases OVID MEDLINE, Cochrane Central Register of Controlled Trials, PubMed, and PsycINFO. Results: Acute doses of CBD were found to reduce anxiety both in animals and humans, without having an anxi- ogenic efect at higher doses. Epidemiological studies tend to support an anxiolytic efect from the consumption of either CBD or THC, as well as whole plant cannabis. Conversely, the available human clinical studies demonstrate a common anxiogenic response to THC (especially at higher doses). Conclusion: Based on current data, cannabinoid therapies (containing primarily CBD) may provide a more suitable treatment for people with pre-existing anxiety or as a potential adjunctive role in managing anxiety or stress-related disorders. However, further research is needed to explore other cannabinoids and phytochemical constituents pre- sent in cannabis (e.g. terpenes) as anxiolytic interventions. Future clinical trials involving patients with anxiety disor- ders are warranted due to the small number of available human studies. Keywords: THC, CBD, Cannabinoid, Cannabis, Anxiety, Anxiolytic, Anxiogenic, Anti-anxiety

Background Cannabis spp. have over 500 phytochemicals docu- mented, including well over 100 cannabinoids, which are unique to the genus [1, 2]. Until recently, cannabis *Correspondence: [email protected] 1 NICM Health Research Institute, Western Sydney University, Locked Bag and its components were largely restricted under inter- 1797, Penrith, Westmead, NSW 2145, Australia national legislation due to the perceived lack of medical Full list of author information is available at the end of the article

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value and the substantial risk of misuse [2]. As a result, administered by itself or concurrently with other can- the pharmacology of most of the cannabinoids are largely nabinoids or terpenes. It has been discussed in the litera- unknown. However, one of the more potent psychoactive ture that CBD, due to its anxiolytic properties, may have compounds, Δ9-tetrahydrocannabinol (THC), has been a protective efect against certain negative psychological extensively isolated, synthesised and studied [3] since it efects from THC [7, 8]. Research has also shown that it was frst isolated in 1964 [4]. Along with the emergence may also be capable of antagonising at least some of the of literature on this compound, there has been a corre- adverse efects related to THC [1, 38]. Recent research sponding increase in the use of cannabis for medical pur- has indicated that when low-dose CBD (4 mg) is com- poses, with the most frequently stated reasons for its use bined with THC the intoxicating efects of THC were being for the management of pain, anxiety and depres- enhanced, while high doses of CBD (400 mg) decreased sion [5]. the same efects [38]. Furthermore, the plethora of chem- Cannabis remains the most commonly consumed illicit ical constituents found in whole cannabis have been drug around the world [6], whilst clinical research is nas- found to be more active than single, purifed phytocan- cent, yet rapidly emerging. Research is urgently required nabinoids [4, 39]. Tis being said, cannabis terpenoids as due to the large variety of cannabis preparations that potential synergistic contributors to the efects of phy- are available on both the licit and illicit drug markets tocannabinoids has not yet been explored in sufcient (depending on jurisdictions) [1]. Furthermore, both com- detail [39]. munity and laboratory-based studies have demonstrated Te plant’s anxiety-modulating action has largely been that the relative quantities of cannabinoids in the plant attributed to a biphasic interaction with the CB1 recep- may directly afect its pharmacological activity when tor. Rey et al. (2012) [40] found that the anxiolytic efects consumed. For example, when taken together with THC, of low doses occur when they interact with the CB1 CBD may potentially ofset some of the adverse efects of receptor on cortical glutamatergic terminals. Conversely, THC, such as memory impairment and paranoia [7, 8]. interaction with the CB1 receptor on the GABAergic ter- It has been demonstrated in rodents that high doses of minals is responsible for anxiogenesis, something which CBD are able to negate some of the anxiogenic response takes place when higher doses are administered. Further, created by THC [9]. the use of a CB1 receptor antagonist has been found to Recreational use of cannabis is commonly reported to fully reverse the efects of THC [41]. However, other non- lead to a feeling of euphoria accompanied by a decrease CB1 receptors are also believed to be involved including in anxiety and an increase in sociability [10]. Conversely, serotonin 5-HT1A receptors [42] and the opioid system it is also frequently reported that cannabis can produce [20, 43, 44]. Tere has also been research in recent years feelings of anxiety, panic, paranoia and psychosis [3, to determine the neural site at which these interactions 11–16]. It has also been demonstrated that changes in take place. Tese studies have largely involved injecting sociability depends on prior exposure and use of canna- THC into various parts of the brain in animal models bis [17]. So why may this contradictory fnding be pre- and observing any anxiolytic or anxiogenic efect [41]; sent? Studies have indicated that the two predominant or by observing the efects of oral doses on the brains of compounds in cannabis: CBD and THC, appear to have individuals under the infuence of THC using functional opposing actions, with the reported anxiolytic efect magnetic resonance imaging (fMRI) [45]. Not surpris- attributed to CBD and anxiogenic outcomes being attrib- ingly, it has also been found that an individual’s history uted to the THC [18]. Nevertheless, a number of more of cannabis use plays a role in the response of an indi- recent publications have shown that this outcome of vidual to cannabis intake [46], something which has been THC is dosage-dependent, with lower dosages having the observed in both animal [20, 42, 43, 47] and human mod- opposite efect. els [48]. Tere is extensive research supporting the biphasic Whilst other papers have reviewed the association of nature of cannabinoids in both anxiety [19–25] (Fig. 1) cannabis with anxiety prevalence [49], or explored the and behavioral responses including motor activity [26– underlying potential anxiolytic or anxiogenic mecha- 30] and aggression [31]. Diferent doses of THC have nisms of action [20, 41–45], or covered the current been found to be biphasic in reward and motor activ- human clinical trial evidence in the area [16], no com- ity [32], and memory and cognition [31, 33]. Whilst prehensive integrated paper exists to date which critically the majority of these studies have been conducted on appraises both the potential anxiolytic and anxiogenic rodents, human studies (covered in detail later) have efects of the plant across these research domains. Tis also provided promising results. Several reports have review seeks to fll this void by compiling a broad over- also found that in animals [34–37], as well as in humans view of the scientifc literature on both the anxiolytic and [37], THC acts diferently according to whether it is angiogenic properties of both whole plant cannabis and Sharpe et al. J Transl Med (2020) 18:374 Page 3 of 21

cross-sectional or longitudinal association between can- nabis use and anxiety, or interventional studies using whole cannabis extracts or isolates (botanically-derived only) for any anxiety disorder, or to test an acute anxi- ogenic or anxiolytic efect. Synthetic cannabinoid ana- logues were excluded from this review. Articles were identifed using the electronic data- bases of OVID MEDLINE, Cochrane Central Register of Controlled Trials, PubMed, and PsycINFO up to Janu- ary 2020, and only included articles in English. No time limits were set. Intervention studies (animal or human) could involve either acute or chronic administration of cannabis-based treatment. Studies testing major cannabi- noids or whole plant interventions were included. Where the composition was unknown, studies where THC was administered via cigarette or inhaler were excluded for the clinical trial portion of this review. In addition, ref- erence lists were searched for additional references. Te main database search was split into three systematic search streams: animal models; epidemiological data; human clinical trials (see Fig. 2). An additional limit was set for epidemiological studies over the past fve years (2016-2020), due to the breadth of current data. Te term ‘signifcant’ was used for a p value of < 0.05. Te following search terms were used to locate ani- mal models as well as epidemiological and intervention studies: “delta 9 thc OR THC OR tetrahydrocannabinol OR delta 9 tetrahydrocannabinol OR delta 9-THC OR D9-THC OR Delta [9] -THC OR Δ9-THC OR CBD OR Fig. 1 Summary of biphasic anxiolytic/anxiogenic efects of cannabis canna* OR terpenes AND “anxi* OR anxiety disorder* OR anxiolytic* OR anti-anxiety OR anxiogenic OR social phobia OR social anxiety OR panic disorder OR post- traumatic stress disorder OR PTSD. Our search revealed a total of 1095 studies with 66 isolates (e.g. THC, CBD, and other phytocannabinoids being relevant for a full review of the articles for poten- and terpenes) in both animals and humans. Tis system- tial inclusion. A fnal review revealed a total of 35 stud- atic review covers animal models, epidemiological data ies eligible for inclusion (17 preclinical, 8 human, and 10 and human clinical trials, concluding with a perspec- epidemiological). tive for industry, clinicians, and the public about current recommendations for medicinal cannabis formulations Results which may provide anxiolytic activity with lesser risk of Epidemiological data anxiogenic efects. Our review of the data revealed 10 studies involving cannabis users consuming whole cannabis preparations or extracts for anxiety (see Tables 1, 2). Included in our Method review were cross-sectional studies with no demographic To provide a comprehensive review of the area, both limitations. Tree studies in particular demonstrated that animal and human studies were sought for inclusion. such use is prevalent, with more than half of the partici- In order to include as many relevant sources as possi- pants in each survey confrming using cannabis for anxi- ble, there were no exclusions based on types of animals ety [50–52]. Further, these studies indicate that there is or models (testing anxiety or mood paradigms) used also a signifcant proportion of people who replaced in the studies. Human studies included in the review some or all prescription medication with cannabis use involved either epidemiological studies exploring the [53, 54]. Te majority of participants were recruited Sharpe et al. J Transl Med (2020) 18:374 Page 4 of 21

online, particularly through social media or through Animal studies medicinal cannabis suppliers. Our initial search returned 1095 articles, with a further Te three cross-sectional studies found that respond- nine studies found through handsearching of the refer- ents reported that they used cannabis medicinally for ences. A total of 17 preclinical studies were found to be anxiety, second only to pain [50, 52, 55], with close to relevant for inclusion (Tables 3 and 4). Te focus of the half of all survey participants stating they use cannabis research concerned primarily CBD and/or THC. for anxiety [50–52, 56]. In a study of 1429 participants, With respect to CBD, both Schier et al. (2012) [60] and Sexton et al. (2016) [50] found that over half (59.8%) of Blessing et al. (2015) [61] concluded that when it was medical users reported using cannabis as an alternative administered acutely, anxiolytic-like efects were only to pharmaceutical prescriptions [50]. Similarly, a US present at low doses, yet has the advantage of not pro- study of 2774 participants found this to be 46% of users ducing anxiogenic efects at higher dose (see Table 3). [53]. Additionally, one study of 2032 people found that Schier et al. (2012) [60] also noted that chronic doses pro- nearly half of the respondents had substituted an anxiety duced mixed results, with both anxiolytic-like and anxio- medication prescribed to them by their physician, with genic-like outcomes being observed. Lee et al. (2017) [62] medical cannabis [56], and 61% indicated that cannabis observed predominantly anxiolytic-like responses in the had completely replaced their prescribed medication. studies analysed, which applied to both acute and chronic Likewise, another study consisting of 1513 participants administration. Ifand & Grotenhermen (2017) [63] con- found similar results, with 71.8% indicating that they cluded that CBD may only be anxiolytic where stress had had reduced their intake of anti-anxiety medications [54] been induced before CBD administration. (Table 2). Tere was also some variance in the results. For exam- In a review of 5085 responses recorded in a smart- ple, Valjient et al. (2002) [21] observed that only the high- phone application, it was found that users of the app est dose of 5.0 mg/kg had an anxiogenic-like efect, and reported signifcantly lower anxiety levels following can- lowest dose of 0.03 mg/kg had an anxiolytic-like efect nabis use [57] (Table 2). Further, only 2.1% experienced in male CD-1 mice. Conversely, Fokos et al. (2010) [64] exacerbated symptoms, while only 4.4% reported no observed the opposite in male Sprague–Dawley rats with change in anxiety symptoms. An Australian study of 1748 the low dose of 0.5 mg/kg producing an anxiogenic-like participants found that fewer than 1% of respondents felt efect and the high dose of 1 mg/kg producing an anxi- that the treated symptom, including anxiety, had wors- olytic-like efect. In McLendon et al.’s (1976) [65] study ened compared to 71 to 92% who felt it had improved of male Rhesus monkeys, all doses from 0.2 mg/kg to [51]. Such results were further confrmed by Turna et al. 1 mg/kg produced an anxiolytic-like response. Con- (2019) [56] where 92% of the 2032 respondents reported versely, Rock et al. (2017) [66] observed an anxiogenic- that cannabis improved their anxiety symptoms. Despite like response for both dosages of 1.0 mg/kg and 10 mg/kg this response, the scores of self-reported questionnaires in male Sprague–Dawley rats. indicate that symptoms remained moderately severe. Tis variance may partly be due to diferent animals In a 3-year longitudinal survey of cannabis use by being studied. While McLendon et al. (1976) [65] used patients with a primary anxiety disorder diagnosis monkeys in their study, this was the only study found to (N = 3723), it was found that remission rates from anxi- do so, with the rest of the reviewed studies using rodents. ety disorders were higher among cannabis nonusers Studies also difered in design, including types of test (Table 2). However, these diferences were not statisti- employed, the size of the apparatus used, dosages admin- cally signifcant in adjusted models [58]. Discrepancies istered, and the route of administration. in responses are further highlighted as men reported experiencing greater headache/migraine relief from Elevated plus‑maze (EPM) medical cannabis than women, despite a larger pro- Braida et al. (2007) [42] injected male Sprague–Dawley portion of women reporting using it for this reason. Of rats with a THC dosage of either 0.015, 0.075 or 0.75 mg/ note also is that women were signifcantly more likely kg and then placed them in the EPM. It was found that than men to report using cannabis to treat anxiety [59]. THC exhibited a dosage-dependent efect with the A summary caveat concerns that the epidemiological highest dosage of THC corresponding to the maxi- data should be considered within the limitation of sur- mum anxiolytic efect. Another approach involved male vey respondents being a ‘captive’ sample who had an Sprague–Dawley rats being administered dosages rang- active interest in cannabis use. ing from 0.075 to 1.5 mg/kg [67]. It was found that even with the addition of a higher dosage compared to the previous study, the maximum anxiolytic efect was still found to occur when the rats were administered 0.75 mg/ Sharpe et al. J Transl Med (2020) 18:374 Page 5 of 21

Fig. 2 Process of identifcation and screening of articles for inclusion kg THC, which supports the idea that depending on the Te next study sought to determine the brain regions dose THC can produce both anxiolytic and anxiogenic involved in producing anxiogenic or anxiolytic efects by responses. Te study by Schramm-Sapyta et al. (2007) injecting THC ranging from 0.001 mg to 0.01 mg directly [68] was unique in that rats were used in their EPM into various parts of the rat brain [41]. Te results indi- instead of mice. Tese male CD rats were also divided cated that in certain regions, diferent dosages produce into two age groups: adolescent and adult. Te rats were opposite efects. For example, when injected into the ven- injected with either 0.5 or 2.5 mg/kg THC. Tey con- tral hippocampus, the lower dose of 0.005 mg produced cluded that while there was a signifcant efect of drug a signifcant anxiolytic-like efect, which switches to an dose on the percentage of time spent in the open arms, anxiogenic-like response when 0.01 mg was injected. In there was no signifcant efect of age on this outcome. At contrast, low doses had no efect when injected into the the lower dose of 0.5 mg/kg though, THC was less anxi- prefrontal cortex, whereas the higher dose of 0.01 mg ogenic in adolescents than in adult rats. produced an anxiolytic like response and 0.025 mg Sharpe et al. J Transl Med (2020) 18:374 Page 6 of 21 Percent Percent of respondents using for anxiety 58.1% 51% 43.7% 52% g/day. ≥ 3 g/day 42% used 1–2 g/day 1 g/day < 14.9% reported using 5–10 times per day every day. 12.2% reported using all day, 25.3% reported using less than once a Dose day is N/A 23% used Dosage 47.6% reported using 1–4 times per day N/A 35% used N/A - cally inhaled (6.4%), Oral (8%), Topical: Topical: cally inhaled (6.4%), Oral (8%), juice (0.5%), Other (0.4%) (0.6%), Fresh - only 29.4% of respond used by routes ents mode of delivery, 21.4% preferred oral 21.4% preferred mode of delivery, 18.5% pre etc.) oils, ingestion (edibles, joints ferred Route of administration Route Inhalation also typi - (84.1%), Concentrates Most frequent route was inhalation (83.4%) route Most frequent and vaporiser suppository, Topical, Oral 47.6% reported vaporising as the preferred 47.6% reported vaporising as the preferred N/A Number of participants 1429 1748 2032 6665 dispensaries in Washington State Washington dispensaries in sional and consumer forums authorized CMP users registered with CMP users registered authorized medicinal cannabis supplier Canadian Tilray included participants 30 over from countries Data source Data Recruited via social media and Cannabis via social media and Cannabis Recruited - via online media and at profes Recruited An online survey all to was disseminated Data were extracted 13 studies that from Data were Epidemiological (part studies of whole cannabis for anxiety 1) 1 Table Study Sexton 2016 [ 50 ] et al. al. 2018 [ 51 ] et al. Lintzeris al. 2019 [ 56 ] et al. Turna 52 ] 2019 [ Kosiba et al. Sharpe et al. J Transl Med (2020) 18:374 Page 7 of 21

Table 2 Epidemiological studies of whole cannabis and cannabis extracts for anxiety (part 2) Study Data Source Number Route of Administration Outcome

Cuttler et al. 2016 [59] Recruited via word-of-mouth 1418 participants Inhaled- (joints, bong, Male: 55.3% and links on advertisements pipe, vaporiser) – (M) Female: 57.2% posted on various websites and 89.8%, (F) 88% Reported feeling less anxious or in Washington State cannabis Oral- (M) 3.9%, (F) 7.9% fearful dispensaries Concentrates- (M) 5.4%, (F) 3.1% Topical- (M & F) 0.4% Other- (M) 0.6%, (F) 0.73% Corroon et al. 2017 [53] Recruited via social media 2774 participants N/A 46% reported using cannabis as a substitute for prescription drugs Piper et al. 2017 [54] New England dispensary mem- 1513 participants N/A 71.8% reduced medication pre- bers scribed for anxiety Corroon et al. 2018 [55] Recruited via social media. 2409 participants N/A Almost 62% of CBD users reported using CBD to treat a medical condition. The top three medical conditions were pain, anxiety, and depression Feingold et al. 2018 [58] Data was drawn from Waves 1 and 3723 participants N/A Remission rates for non-users: 66.0% 2 of the National Epidemiologic Remission rates for users: 52.8%) survey on Alcohol and Related Conditions Cuttler et al. 2018 [57] Data from the cannabis tracking 5085 tracking sessions Inhalation (smoking, vap- 93.5% of sessions recorded decrease app ­StrainprintTM ing, concentrates, dab in anxiety bubbler, dab portable)

Table 3 Reviews of CBD’s anxiolytic properties in rats and mice Study Conclusion de Mello Schier et al. 2012 [60] This reviewed included 17 studies. One study reviewed showed no signifcant efects of high doses of CBD (100 mg/kg) were seen in rats in the Geller-Seifter confict test. In another, a low dose of CBD (10 mg/kg) had anxiolytic efects in rats submitted to the conditioned emotional response. Later studies using the elevated plus maze (EPM) helped to elucidate this contradiction. The authors concluded that anxiolytic efects are only present at low doses Blessing et al. 2015 [61] This review included 32 studies. The authors concluded that overall, preclinical evidence supports systemic CBD as an acute treatment of GAD, SAD, PD, OCD, and PTSD, and suggests that CBD has the advantage of not producing anxiogenic efects at higher dose Lee et al. 2017 [62] This review included 28 studies of both acute and chronic CBD administration on anxiety-like behaviour in male animals. The majority of the listed studies were in Wistar rats, and predominantly, anxiolytic efects were noted Ifand and Grotenhermen 2017 [63] The authors observed in the data that anxiolytic efects in rats were reversed after repeated 14-day administra- tion of CBD. However, this fnding might depend on the used animal model of anxiety or depression and that CBD may only be anxiolytic in subjects where stress had been induced before CBD administration

produced an anxiogenic-like outcome. When injected mice who were injected with 20 mg/kg CBD for 6 weeks into the basolateral amygdala, 0.001 mg THC induced a there was a signifcant decrease in the time spent in the signifcant anxiogenic-like response whereas higher THC open arms [70]. Conversely Zieba et al. (2019) [71] found doses did not afect anxiety behavior. that acute administration of CBD increased time in open In an alternative to the typical rat-model studies above, arms of EPM in male Fmr1 KO mice. Te same mice one study utilised male C57BL/6 JArc mice [69]. When were all given both doses with at least three days between CBD was administered acutely, there was no change in tests. When given the higher dose (20 mg/kg), they were the percentage of time in the open arms or ratio of open- found to spend a longer amount of time in open arms arm entries was observed. Neither was any change in the compared to when they received the lower dose (5 mg/ total number of EPM arm entries. In contrast, Schleicher kg) (p < 0.005 and p < 0.05, respectively) [71]. et al. (2019) [70] found that in male and female C57BL/6J Sharpe et al. J Transl Med (2020) 18:374 Page 8 of 21 like - like the open feld test, all doses of the open feld test, an anxiogenic- THC produced both tests, For like response. no all doses of CBD produced When the combination efect. an THC and CBD was given, of was anxiogenic-like response observed in both the elevated and the open feld plus maze test produced an anxiogenic-like produced and 1 mg/kgefect produced an anxiolytic-like On the efect. in non-stressed other hand, an both doses produced rats, anxiolytic-like response dose an anxiolytic-like response was an anxiolytic-like response When 3.0 mg/kg observed. no response was administered, was seen. like responses like responses an anxiolytic-like response produced an anxiolytic-like produced and the highest dose efect an of (5.0 mg/kg) produced anxiogenic-like All other efect. no efect. doses produced like efect In and plus maze the elevated mg/kg THC 0.5 mg/kgIn rats, stressed No efect was observedNo efect at either mg/kg, 3.0 mg/kg, all doses except At All doses produced anxiolytic- All doses produced All doses produced anxiogenic- All doses produced mg/kg produced dose of 0.3 mg/kgThe produced The lowest dose of 0.03 mg/kg lowest The All does produced an anxiolytic- All does produced Anxiolytic/Anxiogenic efect? (we have looked ta adult have (we mice only in this section) Intraperitoneal Male and Female, mice and Adolescent Adult Intraperitoneal Intraperitoneal Intraperitoneal Intraperitoneal Intraperitoneal Intraperitoneal Intraperitoneal Intravenous Route Elevated plus-maze, Elevated test Open feld (OF) Elevated plus-maze Elevated Elevated plus-maze Elevated Elevated plus-maze Elevated Elevated plus-maze Elevated Light–dark box, (EPM) plus-maze Elevated Light–dark box Light–dark (LD) box Cardiac conditioned response Cardiac Model mg/kg) + CBD (10.0 + 20.0 1.5, 3.0 mg/kg) kg) THC (1.0, 5.0, 10.0 mmg/k) CBD (5.0, 10.0, 20.0 mg/kg) THC THC (0.5, 1.0 mg/kg) CBD (1.0, 50.0 mg/kg) THC (0.015, 0.075, 0.374, 0.75, THC (0.015, 0.075, 0.75 mg/kg) THC (0.5, 2.5 mg/kg) THC (0.3 mg/kg) THC (0.03, 0.1, 1.0, 2.5, 5.0 mg/ THC (0.2, 0.5, 1.0 mg/kg) Compound female C57Bl/6 J mice female CD rats Adolescent and adult male Adolescent Male Sprague–Dawley rats Male C57BL/6JArc mice Male C57BL/6JArc Male Sprague–Dawley rats Male Sprague–Dawley rats Adolescent and adult Male Adolescent Male CD1 mice Male CD-1 mice Male Rhesus monkeys Animal Studies of the anxiogenic/anxiolytic efect of THC and other cannabinoids in animals of the anxiogenic/anxiolytic efectStudies of THC [ 68 ] 2002 [ 20 ] Kasten 2019 [ 72 ] et al. al. 2010 [ 64 ] et al. Fokos al. 2010 [ 69 ] et al. Long al. 2007 [ 67 ] Rubino et al. al. 2007 [ 42 ] Braida et al. al. 2007 et al. Schramm-Sapyta Berrendero and Maldonado Berrendero 21 ] 2002 [ et al. Valjient Acute administration Acute [ 65 ] et al.1976 McLendon 4 Table Study Sharpe et al. J Transl Med (2020) 18:374 Page 9 of 21 - like - like an increase in the time spent an increase in the light and distance was a There Further, ratio. of on an efect towards trend time spent in the inner open but no efect arm in the EPM on the open-arm in the EPM. was a In there the OF test, in the decrease signifcantly time spent in the central zone 15 and on day of the on test the distance ratio decreased THC did not alter one. on day the but decreased SI time, total combined frequency of the social behaviors. the time increased signifcantly spent in the light compart ment and the distance ratio. on the open-armNo efect entry of ratio or percentage time spent on open arms in was an Increase There the EPM. in the time spent central 15. on day of the OF test zone tive to each other (see results each other (see results to tive discussion above) mg/kg CBD produced an 20.0 mg/kg CBD produced On the anxiolytic-like response. no in the OF test, other hand, was observedefect at either dose mg/kg THC, there was THC, there 10 mg/kgAt In CBD (1 mg/kg) the LD test, All dose efects are compara - are All dose efects In both 5.0 and the EPM, Anxiolytic/Anxiogenic efect? Intraperitoneal Intraperitoneal Intraperitoneal Route maze, Open-feld test, maze, Light–dark plus- Elevated box, Social interaction Social Interaction Elevated plus-maze, Elevated Open feld test Model mg/ mg/ mg/ mg/kg, mg/kg, mg/ mg/kg + 5.0 + 20.0 + 5.0 + 20.0 + 20.0 + CBD (1.0 + 5.0 kg, 1.0 kg, 3.0 kg, 3.0 kg, 10.0 kg, 10.0 THC (0.3, 1.0, 3.0, 10.0 mg/kg) CBD 1.0, 5.0, 10.0, 50.0 mg/kg) CBD (5.0, 20.0 mg/kg) THC (1.0, 3.0, 10.0 mg/kg) THC CBD (5.0, 20.0 mg/kg) Compound Male C57BL/6JArc mice Male C57BL/6JArc Male Sprague–Dawley rats Male Fmr1 KO mice KO Male Fmr1 Animal (continued) Chronic administration Chronic 2010 [ 69 ] et al. Long al. 2009 Malone et al. al. 2019 [ 71 ] Zieba et al. 4 Table Study Sharpe et al. J Transl Med (2020) 18:374 Page 10 of 21 like - like ogenic behavior was observedogenic behavior in the light–dark and box but a the open feld test, amount of time decreased was spent in the open arms of the EPM decreased the amount of time decreased on days spent in the light box one and 21, suggesting an well, anxiogenic-like As efect. at a dose of10 mg/kg only, the latency THC increased to but only the light box, enter addition of The one. on day thus increased prior stress anxiogenic efect but anxiogenic-like behavior, administration of CBD abol - ished this efect No changes in anxiolytic/anxi - mg/kg, THC a dose of 1 or 10 mg/kg, At produced A prior stressor Anxiolytic/Anxiogenic efect? Intraperitoneal Intraperitoneal Route Test, Elevated Plus Maze Plus Elevated Test, Light–dark box, Open Field Light–dark Open Field box, Light–dark box Model CBD (20.0 mg/kg) THC (1.0, 10.0 mg/kg) CBD (5.0 mg/kg) Compound mice Male and Female C57BL/6 J Male and Female Male Sprague–Dawley rats Animal (continued) Direct administration into brain Direct administration into 70 ] 2019 [ Schleicher et al. al. 2017 [ 73 ] Rock et al. 4 Table Study Sharpe et al. J Transl Med (2020) 18:374 Page 11 of 21 - - like - like mg microinjected into into 0.005 mg microinjected cortex the prefrontal while 10 mg anxiety behaviour, the percentage THC increase of time and entries the onto open arm, with an consistent anxiolytic- The anxiolytic efect. THC like of 0.01 mg response was also confrmed the by parame analysis of ethological 0.025 mg the anxiolytic At ters. of an was lost in favour efect anxiogenic profle THC the dose of 0.005 mg the increased signifcantly of open arm percentage as the percent time as well This age of open arm entries. was confrmedanxiolytic efect decrease the signifcant by parameter in the ethological Inclosed arm returns. contrast, switch THC seemed to 0.01 mg as an anxiogenic response to in the reduction by shown open arm time and head dips, statisti - although not reaching cal signifcance dose of 0.001 mg induced in the decrease a signifcant of open arm time percentage and head dips a tendency in the open arm decrease to with an consistent entries, anxiogenic-like response. THC doses did not Higher rat anxietyafect in behaviour THC at all doses did the EPM. not change closed arm entries THC at doses of 0.0025 and afect did not signifcantly In hippocampus the ventral the In amygdala the basolateral Anxiolytic/Anxiogenic efect? Microinjection Route Elevated plus-maze Elevated Model - the prefron 0.025 mg into tal cortex, 0.0025, 0.005, the ventral 0.01 mg into 0.001, 0.0025, hippocampus, the 0.005, 0.01 mg into amygdala basolateral THC (0.0025, 0.005, 0.01, Compound Male Sprague–Dawley rats Animal (continued) al. 2008 [ 41 ] Rubino et al. 4 Table Study Sharpe et al. J Transl Med (2020) 18:374 Page 12 of 21

In Long et al.’s (2010) [69] study of chronic adminis- given to male CD1 mice and once again an anxiolytic-like tration, male C57BL/6JArc mice received 21 consecu- response was observed [20]. Tis was done based on the tive daily intraperitoneal injections of either THC (0.3, conclusions of the previous study, and with the inten- 1.0, 3.0 or 10.0 mg/kg) or CBD (1.0, 5.0, 10.0, 50.0 mg/ tion to induce this anxiolytic-like response. Alternative kg). While there was a trend (p = 0.08) towards an efect dosages of 0.3, 1, 3 or 10 mg/kg were also employed [69]. of THC on time spent in the inner open arm, there was Te timeframe also difered, with these given in 21 daily no efect on the open arm entry ratio. When CBD was injections. Tis study implies that there is a clear correla- administered, there was no efect on the open-arm entry tion between increasing dosages of THC and time spent ratio or percentage of time spent on open arms [69]. in the dark area of the LD box. However, there was a similar trend (p = 0.09). towards an In contrast to the other studies, Schramm-Sapyta et al. efect of CBD on time spent in the open arm section clos- (2007) [68] looked at acute THC administration in ado- est to the center zone of the EPM. lescent and adult male CD rats. Te rats received either Like Schleicher et al. (2019) [70], Kasten et al. (2019) 0.5 or 2.5 mg/kg THC. It was observed that the time in [72] also used C57Bl/6 J mice, but also included both the light compartment was signifcantly reduced propor- sexes, and both adults and adolescents in their obser- tionally to increasing dose by THC in both adolescents vations. Te mice were injected with THC (1.0, 5.0 and adults. Conversely, Rock et al. (2017) [73] studied the or 10.0 mg/kg), CBD (5.0, 10.0 or 20.0 mg/kg), and efect of THC chronic administration on male Sprague– THC + CBD (10 mg/kg and 20 mg/kg respectively). Dawley rats using dosages of 1.0 and 10 mg/kg. At the Although there were no trends consistent across all cat- dosages chosen, THC decreased the amount of time egories, they did observe that while there was no sig- spent in the light chamber of the LD box on days one nifcant efect of age there was a signifcant dose-related and 21, suggesting an anxiogenic-like efect both acutely reduction in the time spent in open arms and open arm as well as chronically. Furthermore, at a dose of 10 mg/ entries. Conversely it was observed that there was no kg only, THC increased the latency to enter the light box, interaction between the dose of CBD and the time spent but only on Day 1. Tis latency to enter was increased on the open arms. with the addition of a prior stressor. Long et al. (2010) Another method saw male Sprague–Dawley exposed [69] found that THC given at a high dose of 10 mg/kg to either 10 days of chronic unpredictable stress or no to male C57BL/6JArc mice signifcantly decreased the stressor [64]. After this period, they were injected with time spent in the light compartment. Contrarily, it was either a low (0.5 mg/kg) or a high (1.0 mg/kg) dosage of observed that when the low dose of 1 mg/kg CBD was THC, then being placed in an EPM. It was observed that administered this resulted in a signifcant increase in the in unstressed animals, the rats that were administered time spent in the light compartment. However, when either 0.5 mg/kg or 1 mg/kg THC showed anxiolytic-like 20 mg/kg CBD was given over a period of 6 weeks, no efects. In stressed animals, however, only the high dos- change in anxiety related behaviour was observed [70]. age of THC induced an anxiolytic-like response, whereas the low dosage induced anxiogenic efects. Tese results Open feld (OF) test directly contradict both the idea that THC is anxiolytic Long et al. (2010) [69] tested mice injected with THC at low dosages, and anxiogenic at high dosages at least in an OF test. Te ratio of central to total distance trav- when stress is applied. elled (distance ratio) and the time spent in the central zone were taken as measures of anxiety. It was noted that Light‑dark (LD) box when the maximum dosage of 10 mg/kg was given, there Although the aim of the Valjent et al. (2002) [21] study was a signifcant decrease in the time spent in the central was to determine the efect of THC and nicotine admin- area and a decrease in the distance ratio. Tis was con- istered together, we were able to utilise their results in sistently demonstrated when THC was given daily over this review, as THC was frst administered alone. Tis 21 days, with a signifcantly decreased overall distance involved the acute administration of either 0.03, 0.1, travelled on day 15 and on day 21, the latter of which was 0.3, 1, 2.5 or 5 mg/kg to determine at what dosage THC also observed when doses of 1 mg/kg and 3 mg/kg were would produce a clear anxiolytic-like response. It was given. found that anxiolysis occurred at a dosage of 0.3 mg/ Kasten et al. (2019) [72] found that 5 and 10 mg/kg kg. Tis markedly changed to an anxiogenic efect when doses of THC in adult mice reduced total locomotion. In 5.0 mg/kg was administered and there was no change the 5 mg/kg adult group this was signifcantly correlated in the response relative to vehicle for all other dosages with reduced time in the centre of the open feld indicat- given. Tese fndings were further confrmed when in the ing an anxiogenic-like response. When 10 mg/kg CBD same year the low dosage of 0.3 mg/kg THC was again was given, reduced activity in the adult group was also Sharpe et al. J Transl Med (2020) 18:374 Page 13 of 21

observed, but this was not signifcantly correlated with response (CCR). Te conditioned response is considered anxiety-like metrics. In support of this Long et al. (2010) to be part of the complex of physiological and behav- [69] observed that acute doses of CBD (1 and 50 mg/kg) ioural changes characteristic of anxiety and has been produced an anxiolytic-like efect and Schleicher et al. used to study anxiety in human [65, 76]. Te efect of (2019) [70], who injected male and female C57BL/6 J various dosages of 0.2, 0.5 or 1.0 mg/kg intravenous THC mice over a period of time, found that anxiety behaviour was given. Te results revealed that THC blocked the in the open feld test was not afected. In contrast, Zieba CCR in a dosage dependent manner and this was consist- et al. (2019) [71] found that in their male Fmr1 KO mice ent across trials and across animals. At the lowest dosage acute CBD treatment had no impact on anxiety related tested of 0.2 mg/kg a slight attenuation was consistently parameters in the open feld test [71]. However, they did noticed with a reduction in the conditioned response of 5 fnd that CBD given chronically at 50 mg/kg increased to 6 beats per minute observed. At the next highest dos- the time spent in the central zone of the OF test on day age of 0.5 mg/kg a reduction of 10 to 15 beats per minute 15. was noted for each animal and at the highest dosage of 1 mg/kg, there was a resultant complete block of the CCR Social interaction in every case. Te social interaction test for rodents was frst intro- As detailed in Table 4, our search revealed a range duced by File and Hyde (1978) [74]. In this study experi- of studies of cannabinoids (primarily THC) in anxiety mental manipulation was used to increase anxiety and models beginning in 1976. Research over this period of this was observed to result in a decrease in social interac- 40 + years has revealed conclusions that are inconsistent. tion. Tis test has continued to be used as it is sensitive Generally, the results indicate that at lower dosages an to both anxiolytic and anxiogenic efects [75] and is an anxiolytic response for THC is observed, with the oppo- accepted measure of anxiety-like behaviours. site being true of higher doses (however as indicate above Test male C57BL/6JArc mice and those who had across difering animal modes, this fnding is not always received 0.3, 1, 3 or 10 mg/kg THC were placed in oppo- consistent). site corners of a grey perspex arena to test social inter- action [69]. Mice were allowed to explore freely for Human studies 10 min during which time the authors recorded manu- Of the initial 1095 articles detected in our initial search, ally the frequency and total duration of the active socio- 26 full text articles were assessed for eligibility. Of these, positive behaviours undertaken by the mouse who had 17 met our initial inclusion criteria and an additional fve received the dosage of THC. It was found that while THC were identifed through handsearching of references. Of decreased the combined frequency of the socio-positive these, eight were found to meet inclusion criteria and are behaviours, the total duration of all these behaviours included in this review. remained the same. However, the duration was decreased at 10 mg/kg THC, indicating an anxiogenic-like response Acute human clinical trials at this higher dose. Te anxiogenic properties of isolated THC has have Malone et al. (2009) [9], pre-treated male Sprague– been frmly established in humans and as demonstrated Dawley rats with either vehicle, 5.0 or 20 mg/kg CBD. in Table 5, and no human studies provided any evidence Tese rats were then administered either vehicle, 1.0, 3.0 of anxiolytic efects. However, the dosages administered or 10 mg/kg THC. A signifcant CBD-THC interaction varied widely in the studies described ranging from was observed, as well as a signifcant efect of CBD on the 2.5 mg [48, 77] to 30 mg [78]. In addition there were two total time spent interacting. Te overall trend was that studies which utilised mg/kg [79, 80]. While these two rats treated with a combination of a low dose of CBD and studies are able to be compared more easily with the THC interacted less than rats treated with just the THC. animal studies, this diference in measurement means However, when the dose of CBD was increased, these rats that they are not comparable to the other studies as the interacted more than those treated with just the THC. masses of the participants are not provided. Tis outcome suggests that while CBD is able to negate Evidence of THC’s potential anxiolytic efects in some of the anxiogenic response of THC, higher doses of humans, was frst published in 2004. Te study sample CBD are needed to achieve this. size consisted of 22 healthy individuals who had previ- ously used cannabis, but had never been diagnosed with Cardiac conditioned response (CCR) a cannabis abuse disorder [77]. In a 3-day, double-blind, McLendon et al. (1976) [65] used pairing one of two tones randomised procedure, 22 volunteers received 2.5 or with the delivery of a peripheral electric shock in male 5 mg of THC. Tey were asked to score their feelings Rhesus monkeys to establish the cardiac conditioned using the Visual Analog Scale for anxiety (VAS-A) [81]. Sharpe et al. J Transl Med (2020) 18:374 Page 14 of 21

Te results showed a statistically signifcant increase in Another similar study, used a difering assessment, the VAS-A scores of ‘anxious’. Tis was observed to occur in Subjective Drug Efects Questionnaire (SDEQ) [79]. Ten a dosage-dependent manner, yet there were no statisti- frequent and 10 occasional cannabis users received doses cally signifcant changes in the VAS-A scores for panic. of 0.2, 0.4, and 0.6 mg/kg THC. THC was found to have In a follow up US study, the same methodology was a profound anxiogenic efect, with participants stating applied to people who were frequent users of cannabis that they felt increasingly more tense, jittery and less in [48]. Te researchers aimed to determine if this frequent control as the dose was increased. Karniol et al. (1974) use ofers protection from or tolerance to the efects of [78] also reported a strong anxiogenic reaction as a result THC. Tirty frequent users were compared to 22 healthy of THC administration with subjects expressing that volunteers, who acted as the control. Once again, a cor- the feeling of anxiety sometimes reached a near panic relation between the dosage given and the VAS scores state. Further, four of the fve subjects gave this feel- for anxiety was observed with VAS anxiety scores tran- ing as the maximum grade possible in this study. In this siently increasing in both groups. It was noted that those case, 30 mg of THC was administered. Tis study also who frequently smoked cannabis displayed signifcantly administered various doses of CBD (15.0, 30.0, 60.0 mg) smaller increases in anxiety than controls. to participants. Anxiety was reported by only two of the Converse to the anxiogenic efects of THC, CBD 15 subjects. When CBD was administered with THC, the appears to have the opposite efect. In Bergamaschi et al. anxiogenic efects of the latter were reduced. (2011) [82], participants with social anxiety disorder (SAD) and an additional 12 controls were blindly allo- Discussion cated to receive CBD or placebo 1.5 h before a simulation Data synthesis public speaking test. Te Visual Analogue Mood Scale Te overall pattern of human clinical data supports con- (VAMS), Negative Self-Statement scale, and physiological sistent anxiogenic efects from THC, while CBD shows measures were taken at six time points during the test. a consistent anxiolytic efect. In combination with CBD administration resulted in signifcantly reduced CBD, the anxiogenic efect of THC has been shown to anxiety, cognitive impairment and discomfort, and signif- be decreased. However, further investigation is needed icantly decreased hyper-alertness in anticipatory speech. to categorically afrm this efect. Based on this data, it Further, Crippa et al. (2011) [83], observed regional cer- would imply that cannabis preparations higher in CBD ebral blood fow activity in the brain of participants with and lower in THC cannabis would be most success- SAD who were given CBD or placebo. CBD was found to ful in treating anxiety. However, some survey data does modulate blood fow in the left parahippocampal gyrus, not support this, with a preference for high THC can- hippocampus, and inferior temporal gyrus, and right nabis being of greater interest to consumers for address- posterior cingulate gyrus. In addition, participants who ing afective symptoms. Further to this, only a very small received CBD reported signifcantly lower subjective percentage in surveys reported severe or intolerable side anxiety than those who received a placebo. efects of using cannabis for their symptoms [51]; and Another two studies utilised Spielberger’s State-Trait in general, whole cannabis tends to have a much higher Anxiety Inventory (STAI) to measure anxiety [18, 84]. THC:CBD ratio. Te epidemiological data is in contrast In the frst, participants participated in fve experimen- to the fndings of the clinical trials. tal sessions where they received 0.5 mg/kg THC with the Tese discrepancies could be due to the fact that while STAI being conducted at the start of the frst and last a substantial number of patients cross-sectionally report experimental session [80]. In the second study this was using cannabis and related products to treat anxiety done at baseline and 1,2, and 3 h post administration with symptoms or disorders, it has not been frmly established 10 mg THC [84]. In both cases, an increased STAI score whether this anxiety occurred before or as a result of was noted. Further, it was found that both the STAI and the cannabis usage [16, 85]. As epidemiological research the VAMS scores were signifcantly increased following largely relies on anonymous surveys, the composition of THC intake relative to intake of a placebo. When CBD the cannabis being used is unable to be confrmed. It is was administered alongside THC, this anxiogenic efect known however, that between 1995 and 2015 there has appeared to be reduced [18]. When CBD was given by been a 212% increase in THC content in the marijuana itself, there was no change in the STAI score compared fower [86]. It is also known that plants producing high to the baseline. However, a possible reduction in anxiety levels of THC are incapable of producing much CBD was evidenced in the results of the VAMS anxiety and [86]. Tus, recent studies looking at whole cannabis con- tranquilization subscale [84] Compared with placebo, sumption in theory should provide a relatively reliable CBD administration did not signifcantly change any of source of information regarding the anxiogenic and/or the subject ratings. anxiolytic properties of THC. Our review also highlights Sharpe et al. J Transl Med (2020) 18:374 Page 15 of 21 - - signif CBD produced subjective cantly lower anxiety and modulated in the leftblood fow parahippocampal gyrus, and infe hippocampus, and rior gyrus, temporal right posterior cingulate gyrus resulted in signifcantly in signifcantly resulted - cogni anxiety, reduced impairment and tive discomfort,- and signif hyper- cantly decreased alertness in anticipatory CBD group The speech. controls and the healthy had similar response profles. VAS anxiety scores in anxiety scores VAS However, both groups. users showed frequent in smaller increases anxiety than controls. VAS scores of ‘anxious’ ‘anxious’ of scores VAS and VAS decreased and ‘calm of scores in a dose relaxed’ dependent manner Compared to the placebo to Compared CBD administration THC transiently increased THC transiently increased THC transiently increased THC transiently increased Results Oral Oral Intravenous Intravenous Route CBD (400 mg) CBD (600 mg) THC (2.5, 5.0 mg) THC (2.5, 5.0 mg) Cannabinoid ing, Statistical para - ing, metric mapping anxiety (VAS) anxiety (VAS) anxiety (VAS) - neuroimag Functional Visual Analog Scale for Analog Scale for Visual Visual Analog Scale for Analog Scale for Visual Visual Analog Scale for Analog Scale for Visual Outcome measure Outcome made at fve diferent diferent made at fve time points: 30 min drug inges - before tion, at the time of ingestion and at 60, 75 after and 140 min (or SPECT procedure) after ingestion were taken, followed taken, followed were ingestion of CBD by Pretest or placebo. were measurements made 80 min after ingestion and post- measurements test made 15 and were 35 min after the end of the speech. in 1-3 test days, days, in 1-3 test at least by separated scores VAS 1 week. 60 min recorded were ingestion and before at 10, 80 and 200 min post ingestion. in 1-3 test days, days, in 1-3 test at least by separated scores VAS 1 week. 60 min recorded were ingestion and before at 10, 80 and 200 min post ingestion. Subjective ratings were Subjective ratings were Baseline measurements Baseline measurements Volunteers participated Volunteers Volunteers participated Volunteers Duration - who were given CBD given who were in a dou - or placebo, ble-blinded crossover Regional manner. blood fow cerebral activity in the brain was then compared and an additional 12 blindly were controls receive to allocated CBD or placebo 1.5 h a simulation before public speaking test. six time points At Visual during the test Analogue Mood Scale, Self- and Negative and scale, Statement meas - physiological taken. were ures randomized, placebo- randomized, study over controlled the dose days, three THC of efects related observedwere in 30 users of can - frequent nabis and 22 healthy controls. randomised, and randomised, counterbalanced days, three over study, of the dose efects character THC were ized in healthy indi - in healthy ized viduals who had been cannabis exposed to been but had never with a can - diagnosed nabis abuse disorder. Participants with SAD Participants Participants with SAD Participants In a double-blind, In a double-blind, Methodology = 29, = 9), = 24.8, = 14, female: 21, female: n 21, female: 8), mean age = = age of 24.2 were placed in each were mean age The group. was each group for between 22.9 and 24.6. mean age cannabis, (male: cannabis, n (male: n n who had been cannabis exposed to been but had never with a can - diagnosed nabis abuse disorder 10 males with of a mean 6 males and 6 females 6 males and females 30 frequent users of 30 frequent 22 healthy individuals 22 healthy Participants Studies of cannabinoids for anxiety in humans of cannabinoids for anxiety Studies [ 82 ] al. 2011 [ 83 ] Crippa et al. al. 2011 et al. Bergamaschi D’Souza 2008 [ 48 ] et al. D’Souza 2004 [ 77 ] et al. 5 Table Study Sharpe et al. J Transl Med (2020) 18:374 Page 16 of 21 0.06) for reduction reduction 0.06) for = reactions, with four of with four reactions, subjectsthe fve scoring their anxiety as the level of four. maximum grade anxi - CBD produced in two ogenic efects of the ffteen subjects. When CBD was given THC, the with together of anxiogenic efects reduced. THC were a profound anxiogenic a profound with participantsefect, stating that they felt jittery tense, and more less in control anxiety being noted score. in the STAI CBD did not Though change this signifcantly was a trend there rating, (P in anxiety following CBD administration on anxiety and VAMS the tranquilization subscale. increase in anxiety. in anxiety. increase was particularlyThis when antagonized CBD was administered simultaneously THC provoked strong strong THC provoked THC was found to have have to THC was found THC resulted in increased in increased THC resulted THC produced a large a large THC produced Results Oral Oral Oral Oral Route mg, mg, mg, mg, mg) + 15.0 mg/kg) + 60 + 30.0 CBD (0.5 mg/ + 1.0 + CBD + 30.0 mg (30.0 mg 30.0 mg kg THC (30.0 mg) CBD (15.0, 30.0, 60.0 mg) THC THC (0.2,0.4, 0.6 mg/kg) THC (10.0 mg) CBD (600 mg) THC (0.5 mg/kg) CBD (1.0 mg/kg) THC Cannabinoid panic Questionnaire (SDEQ) Questionnaire Inventory (STAI), VAMS VAMS Inventory (STAI), anxiety and tranquili - zation subscale Inventory (STAI) Reported anxiety and Subjective Drug Efects State-Trait Anxiety State-Trait State-Trait Anxiety State-Trait Outcome measure Outcome production task (asked a 60-sec produce to interval, 10 times), before was repeated and after ingestion. at 55, 95, 155 and At 185 min after inges - tion, subjects were asked questions about and their feelings sensations. administered THC administered or a placebo on four occasions separate at least by separated SDEQ was The 1 week. undertaken 4.5 h after ingestion. on 3 separate occa - on 3 separate each separated sions, a 1-month interval.by THC, CBD or a Either placebo was ingested each session. before participated in fve experimental ses - a by separated sions, minimum interval of 1 week. Pulse rate and a time rate Pulse All 20 subjects were All 20 subjects were Subjects were studied Subjects were Each volunteer Each volunteer Duration - - - male vol healthy dure, assigned were unteers one of eight to experimental groups and in a double-blind received procedure THC, CBD or a mixture of both cannabinoids. a randomised, double a randomised, blind The procedure. participants then flled ques - out a 270 item (SDEQ) tionnaire were administered administered were in a double-blind, randomised manner. magnetic Functional imaging resonance (fMRI) was used to observed the brain while participants images of viewed faces that implicitly induce to intended varying of levels anxiety. a double-blind proce THC, CBD or both dure, cannabinoids together experimen - fve across separated tal sessions, a minimum intervalby of one week. In a double-blind proce THC was administered in THC was administered THC, CBD or a placebo Participants received in received Participants Methodology = 6, female: 2), mean age = aged between 21 and that they 34. 22 stated occa - had previous sional use of cannabis consisting of 5 males aged and 5 females between 21 and 34. reported One group their marijuana use twice did not exceed the per month over The 4 years. past 3 to reported other group, using marijuana twice weeklyor more during the same period. had used cannabis 15 times or less in their with a mean age life of 26.67 of 27. tally stable volunteers tally stable volunteers (male: n n 40 male volunteers 40 male volunteers Two groups, each groups, Two 15 healthy males, who males, 15 healthy 8 physically and men - 8 physically Participants (continued) Karniol 1974 [ 78 ] et al. 79 ] 1976 [ et al. Peters al. 2009 [ 86 ] et al. Fusar-Poli al. 1982 [ 18 ] et al. Zuardi 5 Table Study Sharpe et al. J Transl Med (2020) 18:374 Page 17 of 21

the lack of data from jurisdictions where cannabis is not properties. Kasten et al. (2019) [72] also observed incon- legal, as most of the included studies are based on sur- sistencies across the groups investigated, with adoles- veys by those living in certain states in the US or Canada cent male mice performing diferently to adult male where medicinal use is legal. An important considera- mice, which in turn performed diferently to adolescent tion to note when assessing the epidemiological data is female and adult female mice. Tis clearly shows that age, that many studies are based on self-reported efects from sex and background of exposure may have an impact on participants who are purposively using cannabis for their how an animal or human reacts to THC or CBD inocula- anxiety, and thus due to the sample bias, conclusions tion, something which was found by Cuttler et al’s. (2016) must be tempered. [59] epidemiological survey, where diferent results were In respect to the animal model research, there is strong reported depending on the sex of the person responding. evidence suggesting that an anxiolytic efect occurs after Diferences in methodologies and limitations of data the administration of a small acute dose of CBD [60, 61, provided across the studies reviewed, further reduces 63]. Results however difered depending on whether our ability to draw strong conclusions. Tis includes the CBD was acutely or chronically administered, as well irregularities in doses given, where some studies used as the animal model used. Tis was demonstrated by mg/kg and others mg only, and diferent administra- Rubino et al. (2007) [67] and Schleicher et al. (2019) [70], tion methods being used. Most acute studies using THC who both observed no change in anxiety behaviour in the employ an oral or inhalation route of administration [77]. open feld test, but signifcant changes in behavior in the Oral administration delays the onset of efects by 30 min elevated plus maze. to two hours, produces lower peak plasma levels, and As the present data indicates, no clear conclusion can prolongs the action of the THC compared to the inhaled be drawn from the preclinical studies of acute adminis- or intravenous route [88, 89]. tration of THC. Tis could in part be due to the types of In summary, the human clinical studies using acute animal model being utilised. For example, Onaivi et al. THC consistently produced an anxiogenic efect, while (1990) [19] found that in an elevated plus maze, THC studies using CBD and epidemiological studies of whole induced both in rats and in mice, an increased aversion plant cannabis in anxiety disorders showed an anxiolytic to the open arms of the elevated plus maze; but this efect efect. Tis is surprising as the doses of CBD that have was approximately three times greater in rats than in been shown to have therapeutic efects are far lower than mice. Tus, while the two predominant tests for rodents what is commonly found in cannabis plant matter, such are the elevated plus maze and the light–dark box, the as that which is being used by the majority of partici- results are difcult to compare as rats and mice may pants surveyed in the epidemiological studies [38]. Fur- react diferently to the test paradigm. Tis suggests that thermore, these fndings have not been reliably replicated physiological parameters such as the cardiac conditioned in animal studies, and further larger human RCTs are response used by McLendon et al. (1976) [65] might be a required for stronger validation. more accurate measure as it relies much less on human observation. Development of optimal anxiolytic cannabinoid therapies In humans, research has also shown that the anxiogenic Pharmacological treatment of anxiety relies on our efects of THC are greater among infrequent or non- understanding of the neurobiological interactions users relative to frequent users [16], and high potency responsible [90]. While there are various diferent tar- THC in cannabis products in particular, are thought to gets, the endocannabinoid system has, in recent years, induce the development of psychotic-like symptoms increasingly been attributed with the control of stress, or overt psychosis in vulnerable individuals. Similarly, anxiety and fear. Endocannabinoids appear to modulate intoxication by low-dose CBD has been found to be par- this system as well as the dopamine system, and hypo- ticularly prominent in infrequent cannabis users [38]. thalamo-pituitary-adrenocortical axis [46, 91]. Further, it has been observed in early 1970s research that Tough several classes of synthetic CB receptor ago- individuals who were anxious before receiving it became nists have been developed, these alternatives are high- less anxious under the infuence of cannabis (note that potency ­CB1 receptor activators which elicit pronounced potentially far lower THC preparations would have been psychotropic efects, something which has seen them used). Conversely, non-anxious persons became more recently revoked across most Western countries. THC anxious [87]. In an animal model, Long et al. (2010) on the other hand, is a partial agonist at the CB1 recep- [69] found that diferences were observed amongst mice tor [38, 90], while CBD acts with low-afnity on the CB1 depending on the day in which they were tested, which and CB2 receptors [38]. Cannabis, as a substrate of the suggests that the length of time over which the treat- ­CB1 and ­CB2 receptors in the endocannabinoid system, is ment is given also efects the anxiolytic and anxiogenic therefore a prime substance for investigation. Sharpe et al. J Transl Med (2020) 18:374 Page 18 of 21

However, research has been limited given the contro- Conclusion versial legal history surrounding cannabis. Policies are Te results of this review suggest that there is tentative rapidly evolving, and access to cannabis and cannabi- support based on epidemiological surveys and clinical noid products is increasing worldwide [38], with it now studies showing that whole cannabis and CBD may have being decriminalised or permitted for medical purposes a benefcial role in anxiety disorders (for certain candi- in many countries [92]. In Australia however, this change dates in this population). In contrast, for isolated THC, only took place in 2016. Prior to this it was considered a acute human studies consistently show an anxiogenic schedule 9 drug and so research into its medical use has efect. However, animal studies show that there may be been highly restricted [93]. As such this is still an emerg- potential for THC to be used as an anxiolytic, if given at ing feld. the right dose for the patient, and that this may require With ongoing clinical research into the use of cannabis gradual titration to ameliorate initial anxiogenic efects. for anxiety, it is likely that optimised cannabinoid ratios Further to this, such an approach may be assisted via the of THC and CBD will eventually be better understood. co-administration of CBD, other cannabinoids or ter- Various software programs in use by the general public penes found in the cannabis plant which have yet to be (e.g. Strainprint Technologies, Releaf etc.) may also be of studied substantially. value to researchers tackling this challenge. Apps such Further human studies are needed to establish consist- as these are able to track patient symptoms and collect ency in the results, therapeutic thresholds, and dosage data on the specifc cannabis dosage form, cannabinoid required for cannabinoid therapies to produce an anxio- ratios and particular cannabis products used for certain lytic efect in humans, and further research on cannabi- diseases, conditions or symptomatic relief. noids and terpenes may yield a more optimised anxiolytic Tese two constituents may only be part of the formulation. story, and continuing research into the pharmacological activity of the cannabinoids themselves may reveal that Abbreviations THC and CBD are not the only cannabinoids of clinical CBD: Cannabinol; fMRI: Functional Magnetic Resonance Imaging; LD: Light– interest in anxiety. Notwithstanding the academic appe- dark (box); OF: Open Field (test); SDEQ: Subjective drug efects questionnaire (SDEQ); STAI: State-Trait Anxiety Inventory; THC: Tetrohydrocannabiniol/Δ9- tite for cannabinoid research, an often-overlooked phy- tetrahydrocannabinol; VAMS: Visual analogue mood scale; VAS: Visual analog tochemical class, such as the terpenes/terpenoids, has scale. also shown signifcant anxiolytic action. D-limonene Acknowledgements and linalool, whilst not exclusively found in cannabis, Not applicable. have demonstrated anxiolytic activity; the former via the 5HT1A receptor [4, 94, 95]. As such, specifc chemovars Authors’ contributions LS lead the conception, analysis and performed the bulk of the writing. JS and of cannabis with higher expression of these terpenes may JS guided the initial conceptualisation of the study, supervised the process be of greater clinical interest, particularly when paired throughout, screened articles for inclusion, and along with MdM assisted in with higher CBD concentrations. With such a complex the revision process. AK provided input throughout the conception, design chemistry extent in the Cannabis genus, it is plausible and revision of the work. All authors read and approved the fnal manuscript. that many phytochemicals could be contributing to anxi- Funding olytic activity, likely interacting with numerous receptor J.Sarris is supported by an NHMRC Clinical Research Fellowship (APP112500). types. Further, as previously mentioned, some research Availability of data and materials has shown that the adverse efects of THC, may be dose Not applicable. dependent and are potentially decreased by low doses of Ethics approval and consent to participate CBD [38]. Hence, further research into these interactions Not applicable. would contribute greatly to this area. Lastly, each individual using cannabis is also unique, Consent for publication making the study of pharmacogenomics an important Not applicable. aspect of ongoing cannabis research [96]. Variability in Competing interests cannabinoid receptor genes, transporter genes and phar- No funding was solicited or received for this manuscript. JS and JS conduct cannabis research as university members of the Australia Medicinal Cannabis macokinetic drug metabolism [97], such as that observed Research and Education Collaboration (www.AMCRE​C.org). J. Sarris has in the Cytochrome P450 system, are important factors received consultancy payments from a cannabis manufacturing company for consideration. Further investigation of single nucleo- as an independent scientifc advisor (Australian Natural Therapeutics Group); this is however completely unrelated to this manuscript. He has also received tide polymorphisms (SNPs), in particular of fatty acid either presentation honoraria, travel support, clinical trial grants, book royal- amide hydrolase (FAAH), may potentially afect indi- ties, or independent consultancy payments from: Integria Healthcare & Medi- vidual responses to CBD, and is another worthy research Herb, Pfzer, Scius Health, Key Pharmaceuticals, Fiji Kava, Research Reviews, pathway in the future [96]. Taki Mai, Bioceuticals & Blackmores, Soho-Flordis, Healthworld, HealthEd, Sharpe et al. J Transl Med (2020) 18:374 Page 19 of 21

HealthMasters, Kantar Consulting, Elsevier, Chaminade University, International 17. Babor TF, Mendelson JH, Uhly B, Kuehnle JC. Social efects of marijuana Society for Afective Disorders, Complementary Medicines Australia, SPRIM, use in a recreational setting. Int J Addict. 1978;13(6):947–59. Terry White Chemists, ANS, Society for Medicinal Plant and Natural Product 18. Zuardi AW, Shirakawa I, Finkelfarb E, Karniol IG. Action of cannabidiol on Research, Sanof-Aventis, Omega-3 Centre, the National Health and Medical the anxiety and other efects produced by δ9-THC in normal subjects. Research Council, CR Roper Fellowship. J.Sinclair has sat on the Scientifc Psychopharmacology. 1982;76(3):245–50. Advisory Board for BioCeuticals. LS, AK, and MdM declare that they have no 19. Onaivi ES, Green MR, Martin BR. Pharmacological characteriza- competing interests. tion of cannabinoids in the elevated plus maze. J Pharmacol Exp Ther [Internet]. 1990;253 (3):1002–9. http://ovids​p.ovid.com/ovidw​ Author details eb.cgi?T JS&PAGE refer​ence&D med3&NEWS N&AN 21629​42. 1 NICM Health Research Institute, Western Sydney University, Locked Bag 20. Berrendero= F, Maldonado= R. Involvement= of the opioid= system= in the 1797, Penrith, Westmead, NSW 2145, Australia. 2 Department of Psychiatry, The anxiolytic-like efects induced by Delta (9)-tetrahydrocannabinol. Psy- Melbourne Clinic, Professorial Unit, The University of Melbourne, Melbourne, chopharmacology. 2002;163(1):111–7. Australia. 21. Valjent E, Mitchell JM, Besson M-J, Caboche J, Maldonado R. Behavioural and biochemical evidence for interactions between Delta 9-tet- Received: 22 April 2020 Accepted: 4 September 2020 rahydrocannabinol and nicotine. Br J Pharmacol [Internet]. 2002;135 (2):564–78. http://ovids​p.ovid.com/ovidw​eb.cgi?T JS&PAGE refer​ ence&D med4&NEWS N&AN 11815​392. = = 22. Wachtel= S, ElSohly M, Ross= S, Ambre= J, De Wit H. 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